Novel apoptosis-modulating proteins, DNA encoding the proteins and methods of use thereof

ABSTRACT

The present invention provides a novel family of apoptosis-modulating proteins. Nucleotide and amino acid residue sequences and methods of use thereof are also provided.

[0001] This is a continuation-in-part of U.S. patent application Ser.No. 08/160,067 filed Nov. 30, 1993.

FIELD OF THE INVENTION

[0002] This invention relates to novel proteins withapoptosis-modulating activity, recombinant DNA encoding the proteins,compositions containing the proteins and methods of use thereof.

BACKGROUND OF THE INVENTION

[0003] Apoptosis is a normal physiologic process that leads toindividual cell death. This process of programmed cell death is involvedin a variety of normal and pathogenic biological events and can beinduced by a number of unrelated stimuli. Changes in the biologicalregulation of apoptosis also occur during aging and are responsible formany of the conditions and diseases related to aging. Recent studies ofapoptosis have implied that a common metabolic pathway leading to celldeath may be initiated by a wide variety of signals, including hormones,serum growth factor deprivation, chemotherapeutic agents, ionizingradiation and infection by human immunodeficiency virus (HIV). Wyllie(1980) Nature, 284:555-556; Kanter et al. (1984) Biochem. Biophys. Res.Commun. 118:392-399; Duke and Cohen (1986) Lymphokine Res. 5:289-299;Tomei et al. (1988) Biochem. Biophys. Res. Commun. 155:324-331; Krumanet al. (1991) J. Cell. Physiol. 148:267-273; Ameisen and Capron (1991)Immunology Today 12:102; and Sheppard and Ascher (1992) J. AIDS 5:143.Agents that modulate the biological control of apoptosis thus havetherapeutic utility in a wide variety of conditions.

[0004] Apoptotic cell death is characterized by cellular shrinkage,chromatin condensation, cytoplasmic blebbing, increased membranepermeability and interchromosomal DNA cleavage. Kerr et al. (1992) FASEBJ. 6:2450; and Cohen and Duke (1992) Ann. Rev. Immunol. 10:267. Theblebs, small, membrane-encapsulated spheres that pinch off of thesurface of apoptotic cells, may continue to produce superoxide radicalswhich damage surrounding cell tissue and may be involved in inflammatoryprocesses.

[0005] Bcl-2 was discovered at the common chromosomal translocation sitet(14:18) in follicular lymphomas and results in aberrant over-expressionof bcl-2. Tsujimoto et al. (1984) Science 226:1097-1099; and Cleary etal. (1986) Cell 47:19-28. The normal function of bcl-2 is the preventionof apoptosis; unregulated expression of bcl-2 in B cells is thought tolead to increased numbers of proliferating B cells which may be acritical factor in the development of lymphoma. McDonnell and Korsmeyer(1991) Nature 349:254-256; and, for review see, Edgington (1993)Bio/Tech. 11:787-792. Bcl-2 is also capable of blocking of γirradiation-induced cell death. Sentman et al. (1991) Cell 67:879-888;and Strassen (1991) Cell 67:889-899. It is now known that bcl-2 inhibitsmost types of apoptotic cell death and is thought to function byregulating an antioxidant pathway at sites of free radical generation.Hockenbery et al. (1993) Cell 75:241-251.

[0006] While apoptosis is a normal cellular event, it can also beinduced by pathological conditions and a variety of injuries. Apoptosisis involved in a wide variety of conditions including but not limitedto, cardiovascular disease, cancer regression, immunoregulation, viraldiseases, anemia, neurological disorders, gastrointestinal disorders,including but not limited to, diarrhea and dysentery, diabetes, hairloss, rejection of organ transplants, prostate hypertrophy, obesity,ocular disorders, stress and aging.

[0007] Bcl-2 belongs to a family of proteins some of which have beencloned and sequenced. Williams and Smith (1993) Cell 74:777-779. Allreferences cited herein, both supra and infra, are hereby incorporatedby reference herein.

SUMMARY OF THE INVENTION

[0008] Substantially purified DNA encoding novel bcl-2 homologs, termedcdn-1, cdn-2 and cdn-3, as well as recombinant cells and transgenicanimals expressing the cdn-1 and cdn-2 genes are provided. Thesubstantially purified CDN-1 and CDN-2 proteins and compositions thereofare also provided. Diagnostic and therapeutic methods utilizing the DNAand proteins are also provided. Methods of screening for pharmaceuticalagents that stimulate, as well as pharmaceutical agents that inhibitcdn-1 and cdn-2 activity levels are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 depicts the PCR primers used to isolate the cdn-1 probes.

[0010]FIG. 2 depicts the cdn-1 clones obtained by the methods describedin Example 1.

[0011]FIG. 3 depicts the nucleotide sequence of cdn-1.

[0012]FIG. 4 depicts the results of a Northern blot analysis of multipletissues with probes specific for both bcl-2 and cdn-1.

[0013]FIG. 5 shows the sequence of the cdn-2 cDNA and flanking sequencesand the corresponding predicted amino acid sequence of the cdn-2protein.

[0014]FIG. 6, shows a comparison of N-terminal amino acid sequences ofcdn-1, cdn-2 and known bcl-2 family members.

[0015]FIG. 7 shows the nucleotide sequence of cdn-3.

[0016]FIG. 8 shows the anti-apoptotic effects of cdn-1 and some of itsderivatives in serum-deprivation induced apoptosis of WIL-2 cells.

[0017]FIG. 9 shows anti-apoptotic effects of cdn-1 and some of itsderivatives in FAS-induced apoptosis of WIL-2 cells.

[0018]FIG. 10 shows modulation of apoptosis by cdn-1 and cdn-2 in FL5.12cells.

[0019]FIG. 11 depicts the cdn-1 derivative proteins Δ1, 66 2 and Δ3. TheN-terminal residues are indicated by the arrows. The remainder of thederivative proteins is the same as full-length cdn-1.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention encompasses substantially purifiednucleotide sequences encoding the novel bcl-2 homologs, cdn-1 and cdn-2;and the proteins encoded thereby; compositions comprising cdn-1 andcdn-2 genes and proteins and methods of use of thereof. Note that incopending U.S. patent application Ser. No. 08/160,067, cdn-1 was termedcdi-1; although the name has been changed, the nucleotide sequenceremains identical. The invention further includes recombinant cells andtransgenic animals expressing the cloned cdn-1 or cdn-2 genes. Thenucleotide and predicted amino acid residue sequences of cdn-1 are shownin FIG. 3; and those of cdn-2 are shown in FIG. 5. It has now been foundthat the proteins encoded by the cdn genes are capable of modulatingapoptosis. In a lymphoblastoid cell line, cdn-1 was shown to decreaseFas-mediated apoptosis. In a mouse progenitor B cell line, FL5.12, cdn-2and a derivative of cdn-1 decrease IL-3-induced apoptosis whereas cdn-1slightly increased apoptosis. Thus, depending on the cell type, thederivative of cdn and the method of induction of apoptosis, apoptosiscan be modulated in a highly specific manner by controlling theconcentration of cdns.

[0021] As used herein, “cdns” or “cdn” refers to the nucleic acidmolecules described herein (cdn-1, cdn-2, cdn-3 and derivativesthereof), “the CDNs” or “CDN” refers to the proteins encoded thereby(CDN-1, CDN-2, CDN-3 and derivatives thereof). The present inventionencompasses cdn-1 and cdn-2 nucleotide sequences. The cdn nucleotidesinclude, but are not limited to, the cDNA, genome-derived DNA andsynthetic or semi-synthetic DNA or RNA. The nucleotide sequence of thecdn-1 cDNA with the location of restriction endonuclease sites is shownin FIG. 2. As described in the examples herein, cdn-1 mRNA has beendetected in a variety of human organs and tissues by Northern blotanalysis. These organs include liver; heart; skeletal muscle; lung;kidney; and pancreas as shown in FIG. 3.

[0022] Similarly, cdn-2, cdn cDNA, genomic DNA and synthetic orsemi-synthetic DNAs and RNAs are additional embodiments of the presentinvention. The nucleotide sequence of cdn-2 cDNA, along with thepredicted amino acid sequence of cdn-2 protein and the locations ofrestriction endonuclease recognition sites, is given in FIG. 5. Theexamples presented herein indicate that cdn-1 is on human chromosome 6and that cdn-2 is on human chromosome 20. There is also a member of thefamily cdn-3 which is on human chromosome 11. Fluorescence in situhybridization (FISH) indicated an approximate location of cdn-1 to be at6p21-23. Within this region resides the gene for spinocerebellar ataxiatype 1. Interestingly, apoptosis-has been proposed recently to beinvolved in the related genetic disorder ataxia telangiectasia. Takentogether with the chromosomal localization and the expression of cdn-1in brain tissue, this suggests the possibility that cdn-1/cdn-2 mightrepresent the SCA1 gene locus. It is possible that cdn-2 and cdn-3 arepseudogenes. While these may not be expressed endogenously, they arecapable of expression from a recombinant vector providing theappropriate promoter sequences. Thus, both cdn-2 and cdn-3 genes areencompassed by the present invention as are recombinant constructsthereof and proteins encoded thereby.

[0023] Derivatives of the genes and proteins include any portion of theprotein, or gene encoding the protein, which retains apoptosismodulating activity. FIG. 10 depicts three such derivatives of cdn-1which have been shown to retain apoptosis-modulating activity. Thesederivatives, cdn1-Δ1, cdn1-66 2 and cdn1-66 3, are encompassed by thepresent invention.

[0024] The invention includes modifications to cdn DNA sequences such asdeletions, substitutions and additions particularly in the non-codingregions of genomic DNA. Such changes are useful to facilitate cloningand modify gene expression.

[0025] Various substitutions can be made within the coding region thateither do not alter the amino acid residues encoded or result inconservatively substituted amino acid residues. Nucleotide substitutionsthat do not alter the amino acid residues encoded are useful foroptimizing gene expression in different systems. Suitable substitutionsare known to those of skill in the art and are made, for instance, toreflect preferred codon usage in the particular expression systems.

[0026] The invention encompasses functionally equivalent variants andderivatives of cdns which may enhance, decrease or not significantlyaffect the properties of CDNs. For instance, changes in the DNA sequencethat do not change the encoded amino acid sequence, as well as thosethat result in conservative substitutions of amino acid residues, one ora few amino acid deletions or additions, and substitution of amino acidresidues by amino acid analogs are those which will not significantlyaffect its properties.

[0027] Amino acid residues which can be conservatively substituted forone another include but are not limited to: glycine/alanine;valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamicacid; serine/threonine; lysine/arginine; and phenylalanine/tyrosine. Anyconservative amino acid substitution which does not significantly affectthe properties of CDNs is encompassed by the present invention.

[0028] Techniques for nucleic acid manipulation useful for the practiceof the present invention are described in a variety of references,including but not limited to, Molecular Cloning: A Laboratory Manual,2nd ed., Vol. 1-3, eds. Sambrook et al. Cold Spring Harbor LaboratoryPress (1989); and Current Protocols in Molecular Biology, eds. Ausubelet al., Greene Publishing and Wiley-Interscience: New York (1987) andperiodic updates.

[0029] The invention further embodies a variety of DNA vectors havingcloned therein the cdn nucleotide sequences encoding. Suitable vectorsinclude any known in the art including, but not limited to, those foruse in bacterial, mammalian, yeast and insect expression systems.Specific vectors are known in the art and need not be described indetail herein.

[0030] The vectors may also provide inducible promoters for expressionof the cdns. Inducible promoters are those which do not allowconstitutive expression of the gene but rather, permit expression onlyunder certain circumstances. Such promoters may be induced by a varietyof stimuli including, but not limited to, exposure of a cell containingthe vector to a ligand, metal ion, other chemical or change intemperature.

[0031] These promoters may also be cell-specific, that is, inducibleonly in a particular cell type and often only during a specific periodof time. The promoter may further be cell cycle specific, that is,induced or inducible only during a particular stage in the cell cycle.The promoter may be both cell type specific and cell cycle specific. Anyinducible promoter known in the art is suitable for use in the presentinvention.

[0032] The invention further includes a variety of expression systemstransfected with the vectors. Suitable expression systems include butare not limited to bacterial, mammalian, yeast and insect. Specificexpression systems and the use thereof are known in the art and are notdescribed in detail herein.

[0033] The invention encompasses ex vivo transfection with cdns, inwhich cells removed from animals including man are transfected withvectors encoding CDNs and reintroduced into animals. Suitabletransfected cells include individual cells or cells contained withinwhole tissues. In addition, ex vivo transfection can include thetransfection of cells derived from an animal other than the animal orhuman subject into which the cells are ultimately introduced. Suchgrafts include, but are not limited to, allografts, xenografts, andfetal tissue transplantation.

[0034] Essentially any cell or tissue type can be treated in thismanner. Suitable cells include, but are not limited to, cardiomyocytesand lymphocytes. For instance, lymphocytes, removed, transfected withthe recombinant DNA and reintroduced into an HIV-positive patient mayincrease the half-life of the reintroduced T cells.

[0035] As an example, in treatment of HIV-infected patients by theabove-described method, the white blood cells are removed from thepatient and sorted to yield the CD4⁺ cells. The CD4⁺ cells are thentransfected with a vector encoding CDNs and reintroduced into thepatient. Alternatively, the unsorted lymphocytes can be transfected witha recombinant vector having at least one cdn under the control of acell-specific promoter such that only CD4⁺ cells express the cdn genes.In this case, an ideal promoter would be the CD4 promoter; however, anysuitable CD4⁺ T cell-specific promoter can be used.

[0036] Further, the invention encompasses cells transfected in vivo bythe vectors. Suitable methods of in vivo transfection are known in theart and include, but are not limited to, that described by Zhu et al.(1993) Science 261:209-211. In vivo transfection by cdns may beparticularly useful as a prophylactic treatment for patients sufferingfrom atherosclerosis. Elevated modulation of the levels of CDN couldserve as a prophylaxis for the apoptosis-associated reperfusion damagethat results from cerebral and myocardial infarctions. In these patientswith a high risk of stroke and heart attack, the apoptosis andreperfusion damage associated with arterial obstruction could beprevented or at least mitigated.

[0037] Infarctions are caused by a sudden insufficiency of arterial orvenous blood supply due to emboli, thrombi, or pressure that produces amacroscopic area of necrosis; the heart, brain, spleen, kidney,intestine, lung and testes are likely to be affected. Apoptosis occursto tissues surrounding the infarct upon reperfusion of blood to thearea; thus, modulation of CDN levels, achieved by a biologicalmodifier-induced change in endogenous production or by in vivotransfection, could be effective at reducing the severity of damagecaused by heart attacks and stroke.

[0038] Transgenic animals containing the recombinant DNA vectors arealso encompassed by the invention. Methods of making transgenic animalsare known in the art and need not be described in detail herein. For areview of methods used to make transgenic animals, see, e.g. PCTpublication no. WO 93/04169. Preferably, such animals expressrecombinant cdns under control of a cell-specific and, even morepreferably, a cell cycle specific promoter.

[0039] In another embodiment, diagnostic methods are provided to detectthe expression of cdns either at the protein level or the mRNA level.Any antibody that specifically recognizes CDNs is suitable for use inCDN diagnostics. Abnormal levels of CDNs are likely to be found in thetissues of patients with diseases associated with inappropriateapoptosis; diagnostic methods are therefore useful for detecting andmonitoring biological conditions associated with such apoptosis defects.Detection methods are also useful for monitoring the success ofCDN-related therapies.

[0040] Purification or isolation of CDNs expressed either by therecombinant DNA or from biological sources such as tissues can beaccomplished by any method known in the art. Protein purificationmethods are known in the art. Generally, substantially purified proteinsare those which are free of other, contaminating cellular substances,particularly proteins. Preferably, the purified CDNs are more thaneighty percent pure and most preferably more than ninety-five percentpure. For clinical use as described below, the CDNs are preferablyhighly purified, at least about ninety-nine percent pure, and free ofpyrogens and other contaminants.

[0041] Suitable methods of protein purification are known in the art andinclude, but are not limited to, affinity chromatography, immunoaffinitychromatography, size exclusion chromatography, HPLC and FPLC. Anypurification scheme that does not result in substantial degradation ofthe protein is suitable for use in the present invention.

[0042] The invention also includes the substantially purified CDNshaving the amino acid residue sequences depicted in FIGS. 3 and 5,respectively. The invention encompasses functionally equivalent variantsof CDNs which do not significantly affect their properties and variantswhich retain the same overall amino acid sequence but which haveenhanced or decreased activity. For instance, conservative substitutionsof amino acid residues, one or a few amino acid deletions or additions,and substitution of amino acid residues by amino acid analogs are withinthe scope of the invention.

[0043] Amino acid residues which can be conservatively substituted forone another include but are not limited to: glycine/alanine;valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamicacid; serine/threonine; lysine/arginine; and phenylalanine/tyrosine. Anyconservative amino acid substitution which does not significantly affectthe properties of CDNs is encompassed by the present invention.

[0044] Suitable antibodies are generated by using the CDNs as an antigenor, preferably, peptides encompassing the CDN regions that lacksubstantial homology to the other gene products of the bcl family.Methods of detecting proteins using antibodies and of generatingantibodies using proteins or synthetic peptides are known in the art andare not be described in detail herein.

[0045] CDN protein expression can also be monitored by measuring thelevel of cdn mRNA. Any method for detecting specific mRNA species issuitable for use in this method. This is easily accomplished using thepolymerase chain reaction (PCR). Preferably, the primers chosen for PCRcorrespond to the regions of the cdn genes which lack substantialhomology to other members of the bcl gene family. Alternatively,Northern blots can be utilized to detect cdn mRNA by using probesspecific to cdns. Methods of utilizing PCR and Northern blots are knownin the art and are not described in detail herein.

[0046] Methods of treatment with cdns also include modulating cellularexpression of cdns by increasing or decreasing levels of cdn mRNA orprotein. Suitable methods of increasing cellular expression of cdninclude, but are not limited to, increasing endogenous expression andtransfecting the cells with vectors encoding cdns. Cellular transfectionis discussed above and is known in the art. Suitable indications forincreasing endogenous levels of cdn include, but are not limited to,malignancies and cardiac-specific over-expression. Cardiac specificover-expression is particularly suitable for use in indicationsincluding, but not limited to, patients susceptible to heart disease andin advance of cardiotoxic therapies including, but not limited to,chemotherapies such as adriamycin, so as to offer cardioprotection.

[0047] In addition, increasing endogenous expression of cdns can beaccomplished by exposing the cells to biological modifiers that directlyor indirectly increase levels of CDNs either by increasing expression orby decreasing degradation of cdn mRNA. Suitable biological modifiersinclude, but are not limited to, molecules and other cells. Suitablemolecules include, but are not limited to, drugs, cytokines, smallmolecules, hormones, combinations of interleukins, lectins and otherstimulating agents e.g. PMA, LPS, bispecific antibodies and other agentswhich modify cellular functions or protein expression. Cells are exposedto such biological modifiers at physiologically effectiveconcentrations, and the expression of cdns is measured relative to acontrol not exposed to the biological modifiers. Those biologicalmodifiers which increase expression of cdns relative to the control areselected for further study.

[0048] The invention further encompasses a method of decreasingendogenous levels of cdns. The methods of decreasing endogenous levelsof cdns include, but are not limited to, antisense nucleotide therapyand down-regulation of expression by biological modifiers. Antisensetherapy is known in the art and its application will be apparent to oneof skill in the art.

[0049] Screening for therapeutically effective biological modifiers isdone by exposing the cells to biological modifiers which may directly orindirectly decrease levels of CDNs either by decreasing expression or byincreasing the half-life of cdn mRNA or CDNs. Suitable biologicalmodifiers include, but are not limited to, molecules and other cells.Suitable molecules include, but are not limited to, drugs, cytokines,small molecules, hormones, combinations of interleukins, lectins andother stimulating agents e.g. PMA, LPS, bispecific antibodies and otheragents which modify cellular functions or protein expression. Cells aregrown under conditions known to elicit expression of at least one cdn(preferably cdn-1), exposed to such biological modifiers atphysiologically effective concentrations, and the expression of cdns ismeasured relative to a control not exposed to biological modifiers.Those biological modifiers which decrease the expression of cdnsrelative to a control are selected for further study. Cell viability isalso monitored to ensure that decreased cdn expression is not due tocell death.

[0050] In determining the ability of biological modifiers to modulate(increase or decrease) cdn expression, the levels of endogenousexpression may be measured or the levels of recombinant fusion proteinsunder control of cdn-specific promoter sequences may be measured. Thefusion proteins are encoded by reporter genes.

[0051] Reporter genes are known in the art and include, but are notlimited to chloramphenicol acetyl transferase (CAT) and β-galactosidase.Expression of cdn-1 and -2 can be monitored as described above either byprotein or mRNA levels. Expression of the reporter genes can bemonitored by enzymatic assays, or antibody-based assays, like ELISAs andRIAs, also known in the art. Potential pharmaceutical agents can be anytherapeutic agent or chemical known to the art, or any uncharacterizedcompounds derived from natural sources such as fungal broths and plantextracts. Preferably, suitable pharmaceutical agents are those lackingsubstantial cytotoxicity and carcinogenicity.

[0052] Suitable indications for modulating endogenous levels of cdns areany in which cdn-mediated apoptosis is involved. These include, but arenot limited to, various types of malignancies and other disordersresulting in uncontrolled cell growth such as eczema, or deficiencies innormal programmed cell death such as malignancies, including, but notlimited to, B cell lymphomas.

[0053] The invention also encompasses therapeutic methods andcompositions involving treatment of patients with biological modifiersto increase or decreast expression of cdns. Effective concentrations anddosage regimens may be empirically derived. Such derivations are withinthe skill of those in the art and depend on, for instance, age, weightand gender of the patient and severity of the disease. Alternatively,patients may be directly treated with either native or recombinant CDNs.The CDNs should be substantially pure and free of pyrogens. It ispreferred that the recombinant CDNs be produced in a mammalian cell lineso as to ensure proper glycosylation. CDNs may also be produced in aninsect cell line and will be glycosylated.

[0054] For therapeutic compositions, a therapeutically effective amountof substantially pure CDN is suspended in a physiologically acceptedbuffer including, but not limited to, saline and phosphate bufferedsaline (PBS) and administered to the patient. Preferably administrationis intravenous. Other methods of administration include but are notlimited to, subcutaneous, intraperitoneal, gastrointestinal and directlyto a specific organ, such as intracardiac, for instance, to treat celldeath related to myocardial infarction.

[0055] Suitable buffers and methods of administration are known in theart. The effective concentration of a CDN will need to be determinedempirically and will depend on the type and severity of the disease,disease progression and health of the patient. Such determinations arewithin the skill of one in the art.

[0056] Bcl-2 is thought to function in an antioxidant pathway. Veis etal. (1993) Cell 75:229-240. Therefore, therapy involving CDNs issuitable for use in conditions in which superoxide is involved.Administration of CDNs results in an increased extracellularconcentration of CDNs, which is thought to provide a method of directlyinhibiting superoxide accumulation that may be produced by the blebsassociated with apoptosis. The therapeutic method thus includes, but isnot limited to, inhibiting superoxide mediated cell injury.

[0057] Suitable indications for therapeutic use of CDNs are thoseinvolving free radical mediated cell death and include, but are notlimited to, conditions previously thought to be treatable by superoxidedismutase. Such indications include but are not limited to HIVinfection, autoimmune diseases, cardiomyopathies, neuronal disorders,hepatitis and other liver diseases, osteoporosis, and shock syndromes,including, but not limited to, septicemia.

[0058] Hybridization of cloned cdn DNA to messenger mRNA from variousregions of the brain indicated high levels of expression of cdn-1 ineach of the regions studied (FIG. 8). Therefore, neurological disordersare another area in which therapeutic applications of CDNs may beindicated.

[0059] The following examples are provided to illustrate but not limitthe present invention. Unless otherwise specified, all cloningtechniques were essentially as described by Sambrook et al. (1989) andall reagents were used according to the manufacturer's instructions.

EXAMPLE 1 Identification and Cloning of cdn-1 cDNA

[0060] An amino acid sequence comparison of the six known bcl-2 familymembers (FIG. 6) revealed two regions with considerable sequenceidentity, namely amino acids 144-150 and 191-199. In an attempt toidentify new bcl-2 family members, degenerate PCR primers based onsequences in these regions were designed (FIG. 1) and PCR was performedusing human heart cDNA and human B lymphoblastoid cell line (WIL-2)cDNA. PCR was performed using the Hot Start/Ampliwax technique (PerkinElmer Cetus). The final concentration of the PCR primers and thetemplate cDNA were 4 μM and 0.1-0.2 ng/ml, respectively. The conditionsfor cDNA synthesis were identical to those for first strand cDNAsynthesis of the cDNA library as described below. PCR was performed in aPerkin Elmer Cetus DNA Thermal Cycler according to the method describedby Kiefer et al. (1991) Biochem. Biophys. Res. Commun. 176:219-225,except that the annealing and extension temperatures during the first 10cycles were 36° C. Following PCR, samples were treated with 5 units ofDNA polymerase I, Klenow fragment for 30 min at 37° C. and thenfractionated by electrophoresis on a 7% polyacrylamide, 1×TBE(Tris/borate/EDTA) gel. DNA migrating between 170-210 base pars wasexcised from the gel, passively eluted for 16 hours with gentle shakingin 10 mM Tris-HCl pH 7.5, 1 mM EDTA (TE), purified by passage over anElutip-D column (Schleicher and Schuell), ligated to the pCR-Scriptvector (Stratagene) and transformed into Escherichia coli strainXL1-Blue MRF (Stratagene). Plasmid DNA from transformants (whitecolonies) containing both the heart and WIL-2 PCR products was isolatedusing the Magic Miniprep DNA Purification System (Promega), and the DNAinserts were sequenced by the dideoxy chain termination method accordingto Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467 (USB,Sequenase version 2.0). DNA sequence analysis of the eleven heart PCRproducts revealed two sequences identical to bcl-x (Boise et al. (1993)Cell 74:597-608) and ten other sequences unrelated to the bcl-2 family.

[0061] DNA sequence analyses of the eleven WIL-2 PCR products yieldedone bcl-x sequence, five sequences identical to another bcl-2 familymember, bax (Oldvai et al. (1993) Cell 74:609-619), four unrelatedsequences and one novel bcl-2 related sequence, termed cdn-1. The uniquecdn-1 amino acid sequence encoded by the PCR product is shown in FIG. 6from amino acid 151-190 (top row).

[0062] To isolate the cdn-1 cDNA, a human heart cDNA library (Clontech)and a WIL-2 cDNA library, constructed as described by Zapf et al. (1990)J. Biol. Chem. 265:14892-14898 were screened using the cdn-1 PCR DNAinsert as a probe. The DNA was ³²P-labeled according to the methoddescribed by Feinberg and Vogelstein (1984) Anal. Biochem. 137:266-267and used to screen 150,000 recombinant clones from both librariesaccording to the method described by Kiefer et al. (1991). Eightpositive clones from the WIL-2 cDNA library and two positive clones fromthe heart cDNA library were identified. Four clones from the WIL-2 cDNAlibrary and two from the heart cDNA library were further purified andplasmid DNA containing the cDNA inserts was excised from the λZAPIIvector (Stratagene) (FIG. 2). The two longest clones, W7 (2.1 kb) and W5(2.0 kb) were sequenced and shown to contain the cdn-1 probe sequence,thus confirming their authenticity. The heart cDNAs also encoded cdn-1.

[0063] The W7 DNA sequence along with the deduced amino acid residuesequence is shown in FIG. 2. The deduced amino acid sequence of cdn-1was also aligned for maximum sequence identity with the other bcl-2family members and is shown in FIG. 6. As can be seen, there isconsiderable sequence identity between cdn-1 and other family membersbetween amino acids 100 and 200. Beyond this central region, sequenceconservation falls off sharply. Like bcl-2, cdn-1 appears to be anintracellular protein in that it does not contain a either a hydrophobicsignal peptide or N-linked glycosylation sites. Cdn-1 does contain ahydrophobic C-terminus that is also observed with all bcl-2 familymembers except LMW5-HL, suggesting its site of anti-apoptotic activity,like that of bcl-2, is localized to a membrane bound organelle such asthe mitochondrial membrane, the endoplasmic reticulum or the nuclearmembrane. Hockenbery et al. (1990); Chen-Levy et al. (1989) Mol. Cell.Biol. 9:701-710; Jacobsen et al. (1993) Nature 361:365-369; and Monighanet al. (1992) J. Histochem. Cytochem. 40:1819-1825.

EXAMPLE 2 Northern Blot Analysis of cDNA Clones

[0064] Northern blot analysis was performed according to the methoddescribed by Lehrach et al. (1977) Biochem. 16:4743-4651 and Thomas(1980) Proc. Natl. Acad. Sci. USA 77:5201-5205. In addition, a humanmultiple tissue Northern blot was purchased from Clontech. The codingregions of bcl-2 and cdn-1 cDNAs were labeled by the random primingmethod described by Feinberg and Vogelstein (1984) Anal. Biochem.137:266-267. Hybridization and washing conditions were performedaccording to the methods described by Kiefer et al. (1991).

[0065] The results, presented in FIG. 4 indicate that cdn-1 is expressedin all organs tested (heart, brain, placenta, lung, liver, skeletalmuscle, kidney and pancreas) whereas bcl-2 is not expressed or expressedat only low levels in heart, brain, lung, and liver. Thus, cdn-1 appearsto be more widely expressed throughout human organs than bcl-2 and maybe more important in regulating apoptosis in these tissues.

EXAMPLE 3 Expression of Recombinant cdn-1

[0066] In order to express recombinant cdn-1 in the baculovirus system,the cdn-1 cDNA generated in Example 1 was used to generate a novel cdn-1vector, by a PCR methodology as described in Example 1, using primersfrom the 3′ and 5′ flanking regions of the gene which containrestriction sites to facilitate cloning. The plasmids were sequenced bythe dideoxy terminator method (Sanger et al., 1977) using sequencingkits (USB, Sequenase version 2.0) and internal primers. This was toconfirm that no mutations resulted from PCR.

[0067] A clone was used to generate recombinant viruses by in vivohomologous recombination between the overlapping sequences of theplasmid and AcNPV wild type baculovirus. After 48 hourspost-transfection in insect Spodoptera frugiperda clone 9 (SF9) cells,the recombinant viruses were collected, identified by PCR and furtherpurified. Standard procedures for selection, screening and propagationof recombinant baculovirus were performed (Invitrogen). The molecularmass, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), of the protein produced in the baculovirus system wascompared with the predicted molecular mass of cdn-1 according to theamino-acid sequence.

[0068] In addition, similar clones can be expressed preferably in ayeast intracellular expression system by any method known in the art,including the method described by Barr et al. (1992) Transgenesis ed.JAH Murray, (Wiley and Sons) pp. 55-79.

EXAMPLE 4 Expression of cdn-1 in Mammalian Systems

[0069] The cdn-1 coding sequence was excised from a plasmid generated inExample 1, and introduced into plasmids pCEP7, pREP7 and pcDNA3(Invitrogen) at compatible restriction enzyme sites. pCEP7 was generatedby removing the RSV 3′-LTR of pREP7 with XbaI/Asp718, and substitutingthe CMV promoter from pCEP4 (Invitrogen). 25 μg of each cdn-1-containingplasmid was electroporated into the B lymphoblastoid cell line WIL-2,and stable hygromycin resistant transformants or G418 resistanttransformants (pcDNA3 constructs, FIG. 8) expressing cdn1 were selected.

[0070] The coding region of cdns can also ligated into expressionvectors capable of stably integrating into other cell types includingbut not limited to cardiomyocytes, neural cell lines such as GTI-7 andTNF sensitive cells such as the human colon adenocarcinoma cell lineHT29 so as to provide a variety of assay systems to monitor theregulation of apoptosis by cdn-1.

EXAMPLE 5 Effect of the Anti-Apoptotic Activity of cdn-1 and itsDerivatives in the Wild Type B Lymphoblastoid Cell Line WIL2-729 HF2 andthe Transformed Cell Expressing Excess cdn-1

[0071] 2×10⁵ WIL-2, and WIL-2 cells transformed with a vector encodingcdn-1 as described in Example 4 are grown in RPMI supplemented with 10%fetal bovine serum (FES) for the anti-fas experiment or 0.1% FBS forserum deprivation experiments. In the case of the anti-fas experiment,after washing with fresh medium, the cells were suspended in RPMIsupplemented with 10% FBS, exposed to anti-fas antibodies and thekinetics of cell death in response to an apoptosis inducing agent wereanalyzed by flow cytometry with FACScan. In the case of the serumdeprivation experiment, the WIL-2 cells were resuspended in RPMIsupplemented with 0.1% FBS and apoptosis was monitored according to themethod described by Henderson et al. (1993) Proc. Natl. Acad. Sci. USA90:8479-8483. Other methods of inducing apoptosis include, but are notlimited to, oxygen deprivation in primary cardiac myocytes, NGFwithdrawal, glutathione depletion in the neural cell line GTI-7 or TNFaddition to the HT29 cell line. Apoptosis was assessed by measuring cellshrinkage and permeability to propidium iodide (PI) during their death.In addition, any other method of assessing apoptotic cell death may beused.

[0072]FIG. 8 shows the anti-apoptotic response of various WIL-2transformants to anti-Fas treatment. FIG. 9 shows the anti-apoptoticresponse of various WIL-2 transformants to serum deprivation. In FIG. 8,duplicate wells containing 3×10⁵ cells were incubated with 50 ng/ml ofthe cytocidal anti-Fas antibody for 24 hours. Cell death was thenanalyzed by flow cytometry with FACScan. The proteins expressed fromeach construct are shown beneath the columns. Since many of theconstructs are truncation or deletion variants, the exact amino acidsexpressed are also indicated. As can be seen, all of the transformantshad some protective effect when compared to the control transformantcontaining the pREP7 vector alone. The most apoptosis-resistanttransformant was the cdn-1Δ2 expressing cell line, in which over 90% ofthe cells survived anti-fas treatment. Significant protection was alsoobserved in transformants expressing full length cdn-1 (1-211) andcdn-1Δ1, followed by bcl-2Δ and bcl-2 expressing cell lines.

[0073] Cdn-1Δ1 and cdn-1Δ2 are lacking the N-terminal 59 and 70 aminoacids of the full length cdn-1 molecule, respectively. The observationthat cdn-1Δ2 is more effective at blocking apoptosis than full lengthcdn-1 suggests that smaller, truncated cdn-1 molecules may be potenttherapeutics.

EXAMPLE 6 Determination of Other cdn Genes and Cloning of the cdn-2 Gene

[0074] Southern blot analyses of human genome DNA and a panel ofhuman/rodent somatic cell DNAs indicated that there were at least 3 cdnrelated genes and that they resided in chromosomes 6, 11 and 20.PCR/sequence analysis of the three hybrid DNAs showed that cdn-1 was onchromosome 6 and that two closely related sequences were on chromosome20 (designated cdn-2) and chromosome 11 (designated cdn-3). We havecloned the cdn-2 and cdn-3 genes and sequenced them. Interestingly, bothcdn-2 and cdn-3 do not contain introns and have all of the features ofprocessed genes that have returned to the genome. cdn-3 has a nucleotidedeletion, causing a frame shift and early termination and thus isprobably a pseudogene. Both, however, have promoter elements upstream ofthe repeats CCAAT, TATAAA boxes but are probably not transcribed.(Northern blot analysis with cdn-2 and cdn-3 specified probes.)

[0075] 900,000 clones from a human placenta genomic library in thecosmid vector pWE15 (Stratagene, La Jolla, Calif.) were screened with a950 bp BglII- HindIII cDNA probe containing the entire coding region ofCdn-1. The probe was ³²P-labeled according to the method of Feinberg andVogelstein (1984) Anal. Biochem. 137:266-267. The library was processedand screened under high stringency hybridization and washing conditionsas described by Sambrook et al. (1989) Molecular Cloning, 2nd edition,Cold Spring Harbor Laboratory Press. Ten double positive clones werefurther purified by replating and screening as above. Plasmid DNA waspurified using the Wizard Maxiprep DNA Purification System as describedby the supplier (Promega Corp., Madison, Wis.) and analyzed by EcoRIrestriction enzyme mapping and Southern blotting. The probe used forSouthern blotting and hybridization conditions was the same as above.

[0076] The cosmid clones fell into two groups as judged by EcoRIrestriction analysis and Southern blotting. Cosmid clones (cos) 1-4 and7 displayed one distinct pattern of EcoRI generated DNA fragments andcontained a single 6.5 kb hybridizing EcoRI DNA fragment. Cos2 and Cos9fell into the second group that was characterized by a 5.5 kbhybridizing EcoRI DNA fragment. The 6.5 kb DNA fragment from cos2 andthe 5.5 kb DNA fragment from cos9 were subcloned into pBluescript SK⁻(Stratagene, La Jolla, Calif.) using standard molecular biologicaltechniques (Sambrook et al. as above). Plasmid DNA was isolated and theDNA inserts from two subclones, A4 (from cos2) and C5 (from cos9) weremapped with BamHI, HindIII and EcoRI and analyzed by Southern blottingas described above. Smaller restriction fragments from both clones weresubcloned into M13 sequencing vectors and the DNA sequence wasdetermined.

[0077] The sequence of A4 contains an open reading frame that displays97% amino acid sequence identity with cdn-1. (FIG. 5) The high degree ofsequence identity of this gene with cdn-1 indicates that it is a newcdn-1 related gene and therefore will be called cdn-2. A sequencecomparison of the encoded cdn-2 protein and the other members of thebcl-2 family is shown in FIG. 5. Cdn-2 contains the conserved regions,BH1 and BH2, that are hallmarks of the bcl-2 family, and displays alower overall sequence identity (˜20-30%) to other members, which isalso characteristic of the bcl-2 family. cdn-3 has a frame shift andtherefore does not contain the structural features of cdn-1, cdn-2 orother bcl-2 family members.

EXAMPLE 7 Chromosomal Localization of the cdn-1 and cdn-2 Genes

[0078] Southern blot analysis of a panel of human/rodent somatic cellhybrid DNAs (Panel #2 DNA from the NIGMS, Camden, N.J.) and fluorescentin situ hybridization (FISH) of metaphase chromosomes were used to mapthe cdn genes to human chromosomes. For Southern blotting, 5 μg ofhybrid panel DNA was digested with EcoRI or BamHI/HindIII, fractionatedon 0.8% or 1% agarose gels, transferred to nitrocellulose and hybridizedwith the cdn-1 probe. Hybridization and washing conditions were asdescribed above. For FISH, the cdn-2 subclone, A4, was biotinylatedusing the Bionick Labeling System (Gibco BRL, Gaithersburg, Md.) andhybridized to metaphase chromosomes from normal human fibroblastsaccording to the method described by Viegas-Pequignot in In SituHybridization, A Practical Approach, 1992, ed. D. G. Wilkinson, pp.137-158, IRL Press, Oxford. Probe detection using FITC-conjugated avidinand biotinylated goat anti-avidin was according to the method describedby Pinkel et al. (1988) Proc. Natl. Acad. Sci. USA 85:9138-9142.

[0079] Southern blot analysis showed three hybridizing EcoRI bands inthe human DNA control that were approximately 12 kb, 11 kb and 5.5 kb inlength. Analysis of the somatic cell hybrid DNA indicated that the 12 kbband was in two different samples, NA10629, which contained only humanchromosome 6, and NA07299, which contained both human chromosomes 1 andX and, importantly, a portion of chromosome 6 telomeric to p21. The 11kb band was in NA13140, which contains human chromosome 20. The 5.5 kbhybridizing band was found only in sample NA10927A, which containedhuman chromosome 11. PCR/DNA sequencing analysis of these hybrid DNAsamples using primers for cdn-1 or cdn-2, showed cdn-1 sequences inNA10629 (the chromosome 6-containing hybrid DNA) and NA07299 (thechromosome 1, X and 6pter >p21-containing hybrid DNA), indicating thatthe cdn-1 gene resides on chromosome 6, telomeric to p21. cdn-2sequences were found in NA13140, indicating the cdn-2 gene resides onchromosome 20, and cdn-3 sequences were found in NA10927A, indicatingthe cdn-3 gene resides on chromosome 11.

EXAMPLE 8 Modulation of Apoptosis by cdn-1 and cdn-2 in FL5.12 cells

[0080] FL5.12 is an IL-3-dependent lymphoid progenitor cell line(McKearn et al. (1985) Proc. Natl. Acad. Sci USA 82:7414-7418) that hasbeen shown to undergo apoptosis following withdrawal of IL-3 but isprotected from cell death by overexpression of bcl-2. Nunez et al.(1990) J. Immunol. 144:3602-3610; and Hockenbery et al. (1990) Nature348:334-336. To assess the ability of cdn-1 and cdn-2 to modulateapoptosis, cDNAs encoding cdn-1, cdn-2, two truncated forms of cdn-1(described below) and bcl-2 were ligated into the mammalian expressionvector, pcDNA3 (Invitrogen, San Diego, Calif.) and stably introducedinto the mouse progenitor B lymphocyte cell line FL5.12 byelectroporation and selection in media containing the antibiotic G418.Assays were then performed on bulk transformants as described below.

[0081] The effects of the overexpressed genes on FL5.12 cell viabilitywere examined at various times following withdrawal of IL-3 and areshown in FIG. 10. Cell viability was assessed by propidium iodide (PI)exclusion on a flow cytometer (Becton Dickinson FACScan). Bcl-2expression protected the cells significantly from cell death while cdn-1appeared to enhance cell death when compared to the vector control.Cdn-2 expression conferred a low level of protection from cell death atearlier times but was insignificant at later time points. Interestingly,cdn-1Δ2 gave a moderate level of protection against cell death.Cdn-1-112, a molecule that contains the N-terminal 112 amino acids ofcdn-1, also appeared to partially protect the FL5.12 cells although atlower levels than Bcl-2.

[0082] As shown in Example 7, expression of cdn-1 and cdn-1Δ2 in WIL2cells resulted in increased cell survival in response toanti-Fas-mediated apoptosis and serum withdrawal. Taken together, thesedata suggest that the various cdn molecules are capable of modulatingapoptosis in a positive or negative manner, depending on the cell typeand apoptotic stimuli. Thus, they are effective in preventing cell deathsuch as in the post-ischemic reperfusion tissue damage in the heart orin inducing cell death in cells that have escaped apoptotic control, asis the case in various cancers.

[0083] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

We claim:
 1. A composition comprising a substantially purifiednucleotide sequence encoding a cdn.
 2. The composition according toclaim 1 wherein the nucleotide sequence is derived from genomic DNA. 3.The composition according to claim 1 wherein the cdn is cdn-1.
 4. Thecomposition accoding to claim 3 having the nucleotide sequence depictedin FIG.
 3. 5. The composition according to claim 1 wherein the cdn iscdn-2.
 6. The composition according to claim 5 having the nucleotidesequence depicted in FIG.
 5. 7. A composition comprising a recombinantDNA vector encoding a cdn.
 8. The composition according to claim 7wherein the CDN is CDN-1.
 9. The composition according to claim 8wherein the nucleotide sequence is depicted in FIG.
 3. 10. Thecomposition according to claim 7 wherein the CDN is CDN-2.
 11. Thecomposition according to claim 10 wherein the nucleotide sequence isdepicted in FIG.
 5. 12. The recombinant DNA vector according to claim 7wherein expression of the sequence encoding the cdn under control of aninducible promoter.
 13. A composition comprising a cell transfected witha recombinant DNA vector encoding a cdn.
 14. The composition accordingto claim 13 wherein the CDN-1.
 15. The composition according to claim 14wherein the nucleotide sequence is depicted in FIG.
 3. 16. Thecomposition according to claim 13 wherein the CDN is CDN-2.
 17. Thecomposition according to claim 16 wherein the nucleotide sequence isdepicted in FIG.
 5. 18. A transgenic animal comprising a recombinant DNAvector encoding a CDN.
 19. The transgenic animal according to claim 18wherein the CDN is CDN-1.
 20. The transgenic animal according to claim19 wherein the cdn nucleotide sequence is depicted in FIG.
 3. 21. Thetransgenic animl according to claim 18 wherein the CDN is CDN-2.
 22. Thetransgenic animal according to claim 21 wherein the cdn nucleotidesequence is depicted in FIG.
 5. 23. A composition comprising asubstantially purified CDN protein.
 24. The composition according toclaim 23 wherein the CDN is CDN-1.
 25. The composition according toclaim 24 wherein the nucleotide sequence is depicted in FIG.
 3. 26. Thecomposition according to claim 23 wherein the CDN is CDN-2.
 27. Thecomposition according to claim 26 wherein the nucleotide sequence isdepicted in FIG.
 5. 28. The composition according to claim 23 whereinthe proteins are expressed by recombinant DNA.
 29. The compositionaccording to claim 23 wherein the proteins are native proteins.
 30. Acomposition comprising the proteins according to claim 23 and apharmaceutically acceptable buffer.
 31. The composition according toclaim 30 wherein the proteins are present in therapeutically effectiveamounts.
 32. A composition comprising a monoclonal or polyclonalantibody which recognizes a CDN but is substantially unreactive withother members of the bcl family.
 33. A method of detecting the presenceof a CDN protein in a biological sample comprising the steps of: a)obtaining a cell sample; b) lysing or permeabilizing the cells toantibodies; c) adding anti-cdns-specific antibodies to the cell sample;d) maintaining the cell sample under conditions that allow theantibodies to complex with the cdn; and e) detecting the antibody-cdncomplexes formed.
 34. The method according to claim 33 wherein the CDNis CDN-1.
 35. The method according to claim 34 wherein the nucleotidesequence is depicted in FIG.
 3. 36. The method according to claim 33wherein the CDN is CDN-2.
 37. The method according to claim 36 whereinthe nucleotide sequence is depicted in FIG.
 5. 38. The method accordingto claim 32 wherein the cell sample comprises T cells.
 39. A method fordetecting the expression of a cdn gene in a biological sample comprisingthe steps of identifying the presence of RNA encoding the cdn.
 40. Themethod according to claim 39 wherein the method for identifying thecdn-1 or cdn-2 mRNA is Northern blotting.
 41. A method identifying cdnmRNA comprising the steps of: a) obtaining a cell sample; b) obtainingRNA from the cell sample; c) performing a polymerase chain reaction onthe RNA using primers corresponding to unique regions of the cdn; and d)detecting the presence of products of the polymerase chain reaction. 42.A method of modulating apoptosis-induced cell death comprisingmodulating the endogenous levels of a CDN.
 43. The method according toclaim 40 wherein the CDN is CDN-1.
 44. The method according to claim 43wherein the nucleotide sequence is depicted in FIG.
 3. 45. The methodaccording to claim 42 wherein the CDN is CDN-2.
 46. The method accordingto claim 45 wherein the nucleotide sequence is depicted in FIG.
 5. 47.The method according to claim 41 wherein the CDN is increased bymodulating expression of an endogenous cdn gene.
 48. The methodaccording to claim 46 wherein the cdn gene expressed is encoded by arecombinant gene.
 49. The method according to claim 48 whereinexpression of the gene is under the control of an inducible promoter.50. The method according to claim 49 wherein the cells and transfectedex vivo and further comprising the steps of reintroducing thetransfected cells into the animal.
 51. The method according to claim 50wherein the cells are T lymphocytes.
 52. The method according to claim49 wherein the recombinant gene is transfected into cells in vivo.
 53. Amethod of treating apoptosis in a patient in need thereof comprisingadministering a therapeutically effective amount of CDN.
 54. The methodaccording to claim 53 wherein the CDN is CDN-1.
 55. The method accordingto claim 54 wherein the nucleotide sequence is depicted in FIG.
 3. 56.The method according to claim 53 wherein the CDN is CDN-2.
 57. Themethod according to claim 56 wherein the nucleotide sequence is depictedin FIG.
 5. 58. The method according to claim 53 wherein the CDN isadministered for any indication for which superoxide dismutase has beenindicated.